PROJECT SUMMARY In addition to traditional antimicrobials, targeting host defense pathways is an attractive strategy to limit the adverse effect of bacterial infection. One such pathway that has received considerable attention is autophagy, a process by which cellular constituents are sequestered in a double-membrane vesicle that is subsequently targeted to the lysosome for degradation and recycling. Autophagy is suggested to be critical for cell autonomous defense because many bacterial pathogens are detected within double-membrane vesicles upon internalization. Therefore, it is possible that drugs that target autophagy will be useful in a wide range of diseases downstream of bacterial infections. However, in addition to a direct microbicidal mechanism, autophagy has many substrates and cell type-specific functions that may contribute to the outcome of an infection. Thus, we chose to re-examine the role of autophagy in vivo using two model pathogens ? Salmonella enterica Typhimurium and Staphylococcus aureus. We chose to investigate S. Typhimurium because previous in vitro studies extensively demonstrated that this bacterium is targeted for degradation through autophagy. In contrast, in vitro experiments indicate that S. aureus uses the autophagy machinery for intracellular survival. In preliminary data, we demonstrate that inhibiting autophagy in vivo leads to the opposite outcome that is predicted by the literature. Specifically, autophagy mutants were protected from S. Typhimurium and susceptible to S. aureus. The goal of this proposal is to elucidate the physiological mechanism by which autophagy functions during infection by these two important bacterial pathogens. In Aim 1, we will test a model in which S. Typhimurium recruits the autophagy machinery to repair the Salmonella-containing vacuole (SCV) and evade innate immune sensors. In Aim 2, we will define a novel role for autophagy in regulating the cell surface proteome of the host cells, a function that is critical in limiting damage caused by a pore-forming toxin produced by S. aureus. The results from the proposed experiments will challenge the existing paradigm on the role of autophagy in antimicrobial defense and guide the proper use of drugs that target autophagy.